Fragile X syndrome (“FrX” or “FXS”) is the most common inherited form of mental retardation in males, with reported incidences of 1 in 4000 in males and 1 in 8000 in females. FXS is caused by the absence or a reduced level of the protein encoded by the fragile X mental retardation (“FMR1”) gene; the gene is generally turned off when a CGG DNA repeat with a non-coding portion of the gene is expanded to greater than 200 CGG repeats. The FMR1 gene is on the X chromosome, and is located at Xq27.3. The gene has a length of 38 kb and encodes a 4.4 kb transcript with 17 exons. (O'Donnell and Warren, Annu Rev Neurosci 25:31538 (2002). The gene sequence is publicly available under accession number L29074 in the National Center for Biotechnology Information (NCBI) “Entrez Nucleotide” database via the NCBI website. The fact that females have two alleles for the gene (one on each X chromosome) while males have only one (since they carry one X chromosome and one Y chromosome) accounts for the difference in incidence and severity between the male and female populations.
Fragile X syndrome results from the presence of too many copies of the trinucleotide CGG repeat in the 5′ untranslated region (“UTR”) of the FMR1 gene. Most people carry between about 6 and 40 trinucleotide repeats. In persons with over 200 trinucleotide repeats (known as a “full mutation”), the repeats are generally hypermethylated, and the gene is silenced.
Persons are considered to have a premutation expansion of the FMR1 gene if they have between about 55 to 200 trinucleotide repeats, and to have a full mutation when they have more than 200 repeats. Males and some females with either premutation or full mutation forms of the gene (alleles) are considered to be carriers. Individuals with permutation alleles are at increased risk for developing fragile X-related disorders as adults. Approximately 1 in 5 adult women premutation carriers will experience premature ovarian failure (“POF”) and approximately 40% of men over 50 years of age, and a smaller number of women, who are identified through known fragile x families, will develop the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS) (Jacquemont et al., Lancet Neurol 6:45 (2007)). Both males and females with full mutation forms of the FMR1 gene generally develop features of the child-onset disorder, FXS, although females are usually less affected than are men since they typically have a second allele of the gene without the mutation, which provides some expression of the protein encoded by the FMR1 gene.
Better identification of carriers and earlier identification of infants with Fragile X syndrome could be accomplished by screening of both the general population and of newborns, in particular, for expanded alleles of the gene. (Bailey, D. B. Jr. et al., Ment Retard Dev Disabil Res Rev 10:3-10 (2004)). Early intervention in infants and toddlers with developmental delay (intellectual disability), and associated behavioral problems (e.g., autism), focusing on language, motor, social and cognitive development, results in improved developmental and behavioral outcomes. (See, e.g., Guralnick, M. J. Am J Ment Retard 102:319-45 (1998); Shonkoff, J. P. et al., Handbook of Early Childhood Intervention. New York: Cambridge University Press, (2000); Bailey, D. B. Jr. et al., Ment Retard Dev Disabil Res Rev 10:3-10 (2004)).
No prospective, controlled studies have been carried out, however, that specifically examine the efficacy of early intervention in fragile X syndrome. Early diagnosis would not only permit the study of intervention in such cases, but would allow families to obtain genetic counseling at a time that will make a difference for subsequent pregnancies (Bailey, D. B. Jr. et al., Ment Retard Dev Disabil Res Rev 10:3-10 (2004)). In addition, early diagnosis will become even more important as newer psychopharmacological treatments are developed specifically for FXS (e.g., mGluR5 receptor antagonists; Hagerman, R. J. “Fragile X Syndrome: Diagnosis, Treatment and Research” Baltimore: The Johns Hopkins University Press″ 287-338 (2002); Berry-Kravis, E. et al., Ment Retard Dev Disabil Res Rev 10:42-8 (2004)).
The need for detecting carriers of premutation alleles is increased by recent findings that premutation alleles in females have been found to be associated with premature ovarian failure (Allingham-Hawkins, D. J. et al., Am J Med Genet 83:322-5 (1999); Sullivan, A. K. et al., Hum Reprod 20: 402-12. Epub 2004 (Dec. 17, 2005)). Moreover, a second form of clinical involvement has recently been identified among older male carriers of premutation (FMR1) alleles (Hagerman, R. J. et al., X. Neurology 57:127-30 (2001)), consisting of progressive intention tremor, gait ataxia, Parkinsonism, and autonomic dysfunction; this disorder has been designated “fragile X-associated tremor/ataxia syndrome” (FXTAS). An effective screening tool would reduce the number of missed or incorrect diagnoses for both POF and FXTAS.
It is estimated that at least one-third of all adult male premutation carriers over 50 years of age, who are ascertained through known fragile X families, will develop symptoms of FXTAS, and the penetrance appears to increase with age (Jacquemont, S. et al., JAMA, 291:460-69 (2004)). Given the carrier frequency among males of ˜1/800 (Dombrowski, C. et al., Hum Mol Genet 11:371-8 (2002)), FXTAS appears to be one of the more common single-gene forms of tremor and ataxia among older adult males in the general population.
An effective screening tool for expanded alleles of the FMR1 gene must satisfy several tests: it must be able to reliably detect and size expanded alleles at least through the upper portion of the premutation range; it must be rapid in both primary detection and secondary analysis phases and identify all alleles in the full mutation range for both males and females; it must be able to unambiguously distinguish between females who are homozygous for normal FMR1 alleles (single normal band following polymerase chain reaction (“PCR”); ˜40% of all females) and females with one normal allele and a second, full mutation allele that does not PCR amplify (single normal band, apparent homozygote); this third test has been the greatest impediment to high-throughput screening. Finally, the test should be inexpensive enough for large scale screening.
A number of approaches were considered in trying to develop a screening tool that meets the tests noted above. Strategies considered included fluor-labeled, PCR-based fragment analysis, and Long PCR methods based on bisulfite modification. For example, (“automated”) fluorescent probe-based fragment analysis currently fails all the tests. The method cannot reliably detect premutation alleles throughout the premutation range, particularly in females, due to the rapidly diminishing signal strength of the expanded allele with increasing CGG repeat number; this latter issue requires significant operator involvement for the interpretation of each scan, thus dramatically reducing the throughput of the method. Moreover, the method is too expensive, due to the costs associated with capillary matrix, fluorescent reagents (e.g., primers), and instrument service and overhead.
Genotyping analysis is estimated to cost $15-20 per sample on a thousand sample basis. The method does not provide consistent, positive reads throughout the premutation range, particularly for DNA samples from female carriers. Furthermore, the method requires substantial time for operation and interpretation. Finally, the method does not reliably distinguish between normal homozygous females and the presence of a very large (non-PCR amplifiable) full mutation allele. Therefore, while the fragment analysis approach holds great potential for rapid, automated screening, the technology is not currently sufficiently developed to be used as a screening/testing tool.
PCR approaches based on bisulfite modification of the CGG repeat sequence (conversion of the unmethylated C nucleotides to U nucleotides) (Clark, S. J. et al., Nucleic Acids Res 22:2990-7 (1994)) hold promise for subsequent long PCR amplification, since the bisulfite-modified DNA is both lower in CG-content and of lower sequence symmetry (base pair changes within the CGG repeat element). However, the bisulfite treatment does not reliably preserve sufficient DNA for PCR from small DNA samples, such as those obtained from blood spots, due to the well-known degradation of DNA during the bisulfite conversion process (Grunau, C. et al., Nucleic Acids Res 29:E65-5 (2001)). Thus, whereas some samples can be genotyped using this approach, the method can fail unpredictably due to sample degradation during the bisulfite treatment. This method also requires additional steps in the screening process, including time and additional steps for bisulfite conversion of DNA. Approximately 6 hours are required for a complete conversion, somewhat less time if only partial conversion is required, which adds to the cost through operator time. Finally, it is very important for a successful and reliable analysis that high-quality DNA is used, which is the major pitfall of the bisulfate approach.
Thus, the ability to screen for persons with large numbers of trinucleotide repeats in the FMR1 gene is of considerable importance. The present invention fills these and other needs.